Relevance of plasma lenvatinib concentrations and endogenous urinary cytochrome P450 3A activity biomarkers in clinical practice

Abstract Lenvatinib (LEN), a multitarget tyrosine kinase inhibitor used in various cancer treatments, is mainly metabolized by cytochrome P450 3A (CYP3A) enzymes. The importance of therapeutic drug monitoring (TDM) in patients administered LEN has been proposed. Although some biomarkers of endogenous CYP3A activity have been reported, their utility in dosage adjustments has not been well evaluated. This study investigated the correlation between plasma LEN concentrations and endogenous urinary CYP3A biomarkers in clinical practice. Concentrations of plasma LEN (N = 225) and CYP3A biomarkers (cortisol, 6β‐hydroxycortisol, deoxycholic acid, and 1β‐hydroxydeoxycholic acid) in urine (N = 214) from 20 patients (hepatocellular carcinoma, N = 6; thyroid cancer, N = 3; endometrial cancer, N = 8; and renal cell carcinoma, N = 3) collected for consultation for up to 1 year were evaluated using liquid chromatography–tandem mass spectrometry. Moreover, plasma trough LEN concentrations were predicted using a three‐compartment model with linear elimination for outpatients administered LEN before sample collection. Moderate correlations were observed between the quantified actual concentrations and the predicted trough concentrations of LEN, whereas there was no correlation with endogenous urinary CYP3A biomarkers. The utility of endogenous urinary CYP3A biomarkers could not be determined. However, TDM for outpatients administered orally available medicines may be predicted using a nonlinear mixed effect model (NONMEM). This study investigated the utility of endogenous urinary CYP3A biomarkers for personalized medicine and NONMEM for predicting plasma trough drug concentrations. These findings will provide important information for further clinical investigation and detailed TDM.

1β-hydroxydeoxycholic acid) in urine (N = 214) from 20 patients (hepatocellular carcinoma, N = 6; thyroid cancer, N = 3; endometrial cancer, N = 8; and renal cell carcinoma, N = 3) collected for consultation for up to 1 year were evaluated using liquid chromatography-tandem mass spectrometry.Moreover, plasma trough LEN concentrations were predicted using a three-compartment model with linear elimination for outpatients administered LEN before sample collection.Moderate correlations were observed between the quantified actual concentrations and the predicted trough concentrations of LEN, whereas there was no correlation with endogenous urinary CYP3A biomarkers.The utility of endogenous urinary CYP3A biomarkers could not be determined.However, TDM for outpatients administered orally available medicines may be predicted using a nonlinear mixed effect model (NONMEM).This study investigated the utility of endogenous urinary CYP3A biomarkers for personalized medicine and NONMEM for predicting plasma trough drug concentrations.These findings will provide important information for further clinical investigation and detailed TDM.

| INTRODUC TI ON
Lenvatinib (LEN), an orally available multitargeting tyrosine kinase inhibitor, is used as an anticancer drug in patients with various cancers, including hepatocellular carcinoma, 1,2 thyroid cancer, 3 thymic carcinoma, 4 endometrial cancer, 5 and renal cell carcinoma. 5The approved initial dose varies for cancer types.Namely, LEN dosages of 8 or 12 mg/day for hepatocellular carcinoma, 20 mg/day for thyroid and endometrial cancer, and 24 mg/day for thymic and renal cell carcinoma are required for cancer therapy.
7][8] For instance, the optimal plasma trough LEN concentration in patients with thyroid cancer is expected to be from 42 to 88 ng/mL for optimal response, whereas drug toxicity may occur at more than 88 ng/ mL. 7In contrast, the expected target trough concentration of LEN in patients with hepatocellular carcinoma was reported to be between 36.8 and 71.4 ng/mL for maintaining disease control status with reduced toxicity. 8Various adverse effects caused by LEN, including hypertension, diarrhea, proteinuria, and fatigue, are known to occur frequently, resulting in dosage reduction or drug suspension. 9,10However, determining the optimal LEN dose prior to drug treatment is difficult.
LEN is mainly metabolized to N-oxide and O-desmethyl LEN by CYP3A4. 21Interestingly, although the metabolism of numerous drugs overlaps with that of CYP3A4 and CYP3A5, LEN metabolism via CYP3A5 does not occur in vitro. 22The effect of CYP3A4 activity on plasma LEN concentration has been reported. 23,24The dose-adjusted plasma LEN concentrations in patients carrying the CYP3A4*1G allele were significantly lower than those in patients carrying wild-type CYP3A4. 24Thus, LEN dosage adjustment, considering CYP3A4 enzymatic activity, may provide a more precise anticancer therapy.
This study aimed to investigate the clinical usefulness of urinary endogenous CYP3A biomarkers by evaluating the correlation between plasma LEN concentrations and urinary biomarker concentrations.Moreover, a nonlinear mixed effect model (NONMEM) was applied to trough LEN concentrations in the plasma without any additional burden on the patients.

| Subjects
Inpatients and outpatients who started administration of LEN for treating hepatocellular carcinoma, thyroid cancer, endometrial cancer, or renal cell carcinoma between April 2022 and September 2023 were recruited from Tohoku University Hospital (Sendai, Japan).The characteristics of the participants are summarized in Table 1.Plasma samples were collected after taking LEN but not trough point and immediately stored at −80°C until use.Urine samples were collected biomarkers, cytochrome P450 3A, lenvatinib, therapeutic drug monitoring on the same day as blood samples, within 60 min before or after blood sampling.Plasma and urine samples from the same patients were collected continuously for up to 1 year.

| Quantification of plasma LEN concentrations
The plasma LEN concentrations were quantified using previously validated, conventional methods. 2 Briefly, 160 μL of acetonitrilemethanol (9:1, v/v) solution containing 0.8 ng of lenvatinib-2 H 5 was added to 40 μL of the plasma samples.The mixture was vortexed and centrifuged at 14 000 g for 10 min at 4°C.After centrifugation, 2 μL of supernatant was injected into the LC-MS/MS for quantification.
An LC-MS/MS system was used in positive ion detection mode at the electrospray ionization interface (LCMS8050; Shimadzu, Kyoto, Japan).For separation, the NexeraX2 ultra-high-performance liquid chromatography system (Shimadzu) was used with a YMC-Triart C18 metal-free column (2.1 mm i.d.× 50 mm, 3 μm; YMC, Kyoto, Japan) maintained at 40°C.Mobile phases were prepared using deionized water containing 20 mM ammonium formate (pH 3.6) as eluent A and methanol containing 20 mM ammonium formate (pH 3.6) as eluent B. The gradient program was as follows: elution was initiated using 5% eluent B for 1 min, followed by a linear gradient of 100% eluent B for 1-3 min.A linear gradient of 100% eluent B was maintained for 1 min and then immediately returned to the initial conditions, which were maintained for 1 min until the end of the run, at a flow rate of 0.45 mL/min.Quantification analyses were performed in the selected reaction-monitoring mode where ions transitioning from the precursor into product ions were monitored: mass-to-charge ratio (m/z) 427.2 → 370.0 for LEN (Q1 pre-bias, −14 V; collision energy, −27 V; and Q3 pre-bias, −28 V) and m/z 432.2 → 370.1 for lenvatinib-2 H 5 (Q1 pre-bias, −25 V; collision energy, −28 V; and Q3 pre-bias, −26 V).The optimized mass spectrometry settings were as follows: probe voltage, 4000 V; interface temperature, 300°C; desolvation line temperature, 250°C; block heater temperature, 400°C; nebulizing gas flow, 3 L/min; drying gas flow, 10 L/min; and heating gas flow, 10 L/min.Standard curves for LEN were constructed within the range of 1-4000 ng/mL using metabolite standards.The lower limit of quantification was set at 1 ng/mL, 3 considering previously reported plasma trough LEN concentrations. 7,8

| Plasma LEN concentration prediction
The chronological plasma LEN concentrations were predicted using the NONMEM system (ICON Clinical Research, PA, USA).First, each parameter from a previously published three-compartment model with linear elimination that utilized population pharmacokinetics data from 15 clinical studies was set in a text file. 35Specifically, plasma trough LEN concentrations were predicted by considering the following information: body weight, alkaline phosphatase value, whether any CYP3A4 inhibitors were co-administered, LEN dosage, LEN-taking time, and LEN concentrations in plasma.Each dataset was constructed with medication status and blood LEN concentration for 7 days (more than four to five half-lives) before and after blood collection.The approximate daily dosing times were confirmed, and the relationship with blood sampling time was summarized and used for plasma LEN concentration prediction.

| Urinary CYP3A biomarker quantification
The concentrations of urinary biomarkers (1β-OHDCA, DCA, 6β-OHC, and C) were quantified as previously validated methods. 34iefly, 100 μL urine was deproteinized by mixing with 900 μL acetonitrile containing internal standards ( and the concentration per dose ratio when analyzing all patients (R 2 = .2795and .2089,respectively).Furthermore, the correlations were moderate or strong (generally defined as .49≤ R 2 ), except for endometrial cancer, when the results were classified according to cancer type (Figure 2).
In almost all samples, urinary CYP3A biomarkers were quantified within a calibration standard concentrations range of values of the samples for which both parameters were available for comparison.There was no correlation between the ratio of CYP3A biomarker concentrations and predicted plasma trough LEN concentrations (Figure 4).

| DISCUSS ION
3][4][5]21 A therapeutic window for LEN treatment has been suggested for some cancer patients; therefore, chronological monitoring of LEN concentrations in the plasma is important. 7,8Endogenous CYP3A biomarkers, which have been used to predict enzymatic CYP3A activity and dosage adjustment, may be applicable for the drug therapy management of LEN by considering its metabolism.This study evaluated the efficacies of measuring endogenous urinary CYP3A biomarkers (C, 6β-OHC, DCA, and 1β-OHDCA) compared to plasma LEN concentrations.
Moreover, plasma trough LEN concentrations were predicted using NONMEM, the gold standard software for population pharmacokinetics and pharmacokinetic-pharmacodynamic modeling. 39is study protocol was designed to be noninvasive and to reduce the burden on outpatients.We focused on the NONMEM program and endogenous urinary CYP3A biomarkers.5][46] Moreover, the development of orally available drugs for cancer therapy contributes to this need.Plasma LEN concentrations have been quantified in several studies involving patients with hepatocellular carcinoma and thyroid cancer, and a therapeutic window for trough LEN concentrations in hepatocellular cancer treatment has been proposed. 7,8wever, accurately determining plasma trough LEN concentrations may be challenging due to the use of 5 days on/2 days off administration in some patients. 47,48In the present study, plasma LEN concentration parameters, including patients with various cancer types, dosages, and usage, were used to predict plasma trough LEN concentrations, resulting in a moderate correlation between the plasma  LEN concentration at the time of sampling and the predicted plasma trough LEN concentration.Thus, NONMEM may be useful in further clinical studies and future clinical applications.Interestingly, high correlation was observed in the patients with renal cell carcinoma probably because only three patients were administrated LEN everyday but not on the 5 days on/2 days off regimen.In contrast, the correlations in patients with endometrial cancer were relatively weak, possibly because no data from patients with endometrial cancer were included in the parameters used for prediction by NONMEM.
The three graphs for all cancer patients, hepatocellular carcinoma, and endometrial cancer appeared to show two correlations in one graph.This may be due to factors such as differences in liver function derived from cancer type and the lag between LEN dosing times (morning or evening) and blood collection times.Taken together, these findings suggest that optimizing parameters for each cancer type including endometrial cancer, for which NONMEM parameters have not been explored, may be useful in personalized medicine.
The utility of endogenous CYP3A biomarkers was evaluated by comparing plasma LEN concentrations.In this study, the ratios of 6β-OHC/C and 1β-OHDCA/DCA were evaluated for their correlation with plasma LEN concentrations.The relevance between them has not been evaluated until now.Thus, this study can potentially compare them to identify the best biomarker.The concentrations of all analytes and the correlation between the substrate and metabolite indicated a similar trend compared with previous research on 18 healthy participants. 34However, intra-and interratio differences in patients largely varied, and unfortunately, no correlations were observed.In some patients, the fluctuation ranges were different between biomarkers.Specific factors impacting their levels, including the renal clearance of C, may affect these differences, but the values could not be evaluated in this study.Previously reported CYP3A activity biomarkers have been identified in strictly managed clinical studies. 25,26,30,32For instance, the ratio alteration between 1β-OHDCA and DCA was recognized under the condition of taking strong CYP3A inhibitors and urine sampling simultaneously on the same day. 26However, these biomarkers are difficult to manage in clinical practice; therefore, the clinical application of endogenous urinary biomarkers may be challenging.
2][33] Compared to other biomarkers, 4β-hydroxycholesterol has a relatively long half-life. 31For patients with stable conditions without any change in medication, 4β-hydroxycholesterol could be better for predicting CYP3A activities, but it is less important to investigate this biomarker in clinical practice compared to other biomarkers with short half-lives because dosage adjustments should already be completed.The difficulty in predicting enzymatic activity may be specific to the CYP isoforms.
0][51] This biomarker can detect whether a patient is a CYP2D6-poor metabolizer or not.The usefulness of CYP biomarkers may depend on CYP isoforms as the intra-and inter-variety of CYP3A is larger than that of CYP2D6. 52,53Therefore, it may be difficult to predict CYP3A activity although further investigations are required to confirm its utility.
In summary, clinically relevant concentrations of plasma LEN and endogenous urinary CYP3A activity biomarkers were investigated.
The utility of NONMEM for clinical studies and future clinical applications was confirmed, whereas endogenous urinary CYP3A biomarkers are difficult to apply clinically.More accurate plasma trough LEN concentrations using NONMEM would be needed to reveal the actual timing of taking LEN and blood sampling and to optimize parameters for each cancer type.However, large amounts of patient data must be collected.Nevertheless, the utility of TDM for LEN should be investigated in more detail in future studies because the correlation between LEN concentrations and the efficacy and safety of LEN therapy could not be accurately evaluated during this study period despite monitoring LEN dosage reduction in several patients.
CYP3A biomarkers may not be clinically useful; however, there is no consensus due to small sample sizes.For example, a more restricted study design is important for accurate evaluation, such as the correlation between the blood trough concentration of tacrolimus, which is primarily metabolized by CYP3A, 17

ACK N OWLED G M ENTS
We are deeply grateful to all participating patients who provided plasma and urine samples.We also extend our gratitude to the clini-

F I G U R E 1
Metabolic pathways from cortisol (C) to 6β-hydroxycortisol (A) and deoxycholic acid to 1β-hydroxydeoxycholic acid (B) catalyzed by cytochrome P450 3A.TA B L E 1 Background characteristics and therapy schedules for each participant.

F I G U R E 2
Correlation with plasma lenvatinib (LEN) concentrations for all patients and patients with each cancer type.(A) Predicted plasma trough LEN concentrations and (B) predicted plasma trough LEN concentrations per LEN dose.F I G U R E 4 Correlation between 6β-hydroxycortisol (6β-OHC)/cortisol (C) ratio and predicted plasma trough lenvatinib (LEN) concentrations (A) and predicted plasma trough LEN concentrations per dose (B).Correlation between 1β-hydroxydeoxycholic acid (1β-OHDCA)/deoxycholic acid (DCA) ratio and predicted plasma trough LEN concentrations (C) and predicted plasma trough LEN concentrations per dose (D).
and the concentrations of endogenous CYP3A biomarkers in urine, collected simultaneously.The LEN in urine could affect CYP3A biomarkers, but urine concentrations were not evaluated in this study.Therefore, further studies are required to determine the utility of CYP3A biomarkers accurately.AUTH O R CO NTR I B UTI O N S Masaki Kumondai: Conceptualization, methodology, analysis, investigation, data curation, writing-original draft, visualization, and project administration.Reika Ogawa: Analysis, investigation, data curation, and writing-review & editing.Nagomi Hayashi: Analysis, data curation, and writing-review & editing.Yurika Ishida: Investigation, data curation, and writing-review & editing.Hanae Oshikiri: Investigation, data curation, and writing-review & editing.Yuji Sato: Investigation, data curation, and writing-review & editing.Masafumi Kikuchi: Methodology, analysis, and writing-review & editing.Yu Sato: Methodology and writing-review & editing.Toshihiro Sato: Methodology and writing-review & editing.Masamitsu Maekawa: Methodology, writing-review & editing, resources, and supervision.Nariyasu Mano: Methodology, writing-review & editing, resources, and supervision.
staff at the Department of Pharmaceutical Sciences, Division of Gastroenterology, Department of Obstetrics and Gynecology, Department of Breast and Endocrine Surgery, Division of Urology, and Department of Hematology and Rheumatology at Tohoku University Hospital.We thank Editage (www.edita ge.com) for the English language editing.